Correlative imaging of the murine hind limb vasculature and muscle tissue by MicroCT and light microscopy

Correlative Imaging of the Murine Hind Limb Vasculature and Muscle Tissue by MicroCT and Light Microscopy 1Scientific RepoRts | 7 41842 | DOI 10 1038/srep41842 www nature com/scientificreports Correla[.]

Correlative Imaging of the Murine Hind Limb Vasculature and Muscle Tissue by MicroCT and Light

Microscopy

Laura Schaad1,2,*, Ruslan Hlushchuk1,*, Sébastien Barré1, Roberto Gianni-Barrera3, David Haberthür1, Andrea Banfi3 & Valentin Djonov1

A detailed vascular visualization and adequate quantification is essential for the proper assessment

of novel angiomodulating strategies Here, we introduce an ex vivo micro-computed tomography

(microCT)-based imaging approach for the 3D visualization of the entire vasculature down to the capillary level and rapid estimation of the vascular volume and vessel size distribution After perfusion with μAngiofil®, a novel polymerizing contrast agent, low- and high-resolution scans (voxel side length: 2.58–0.66 μm) of the entire vasculature were acquired Based on the microCT data, sites

of interest were defined and samples further processed for correlative morphology The solidified, autofluorescent μAngiofil® remained in the vasculature and allowed co-registering of the histological sections with the corresponding microCT-stack The perfusion efficiency of μAngiofil® was validated based on lectin-stained histological sections: 98 ± 0.5% of the blood vessels were μAngiofil®-positive, whereas 93 ± 2.6% were lectin-positive By applying this approach we analyzed the angiogenesis induced by the cell-based delivery of a controlled VEGF dose Vascular density increased by 426% mainly through the augmentation of medium-sized vessels (20–40 μm) The introduced correlative and quantitative imaging approach is highly reproducible and allows a detailed 3D characterization of the vasculature and muscle tissue Combined with histology, a broad range of complementary structural information can be obtained.

Occlusive vascular disorders, such as peripheral artery disease (PAD) and critical limb ischemia, are major global health issues, causing vascular morbidity and mortality In 2010, a total of 202 million patients throughout the world were suffering from PAD1 Therapeutic angiogenesis is a potential treatment strategy to modulate the microcirculation and therefore ameliorate ischemic conditions in those patients Particularly, vascular endothe-lial growth factors (VEGFs), being key regulators of angiogenesis, have been extensively studied as potential drug treatment, although with limited success2–5 Recent studies have focused on investigating vascular alterations caused by the application or discontinuation of angiomodulating therapies onto diseased and healthy tissues6,7 The murine hind limb is one of the most widely used preclinical models to study therapeutic angiogenesis This is mainly due to the anatomical similarities with the human limb and its relatively simple microvascular architecture with most capillaries running in parallel to the muscle fibres The current gold standard for quantify-ing skeletal muscle’s microvasculature is assessquantify-ing capillary density and capillary-to-fiber ratio based on histolog-ical cross-sections However, histologhistolog-ical approaches have several significant limitations Besides shrinkage and tissue distortion occurring during sample preparation, this technique does not provide any information about the three-dimensional (3D) vascular architecture or the vascular pattern, unless serial sectioning is performed Serial sectioning, however, is very laborious, time-consuming, and the precise alignment is often delicate8,9 Vascular corrosion casts examined by scanning electron microscopy is another tool commonly used to study the micro-vasculature10 Although it provides images from superficial vessels, deeper vessels inside the sample cannot be visualized and quantitative information can barely be extracted Similar limitations also apply to earlier studies

1Institute of Anatomy, University of Bern, Baltzerstrasse 2, 3012 Bern, Switzerland 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland 3Department of Biomedicine, University Hospital Basel, Hebelstrasse 20, 4031 Basel, Switzerland *These authors contributed equally to this work Correspondence and requests for materials should be addressed to V.D (email: djonov@ana.unibe.ch)

received: 03 October 2016

accepted: 22 December 2016

Published: 07 February 2017

OPEN

investigating the skeletal muscle microcirculation by transillumination, and thus being restricted to thin muscles (e.g., murine spinotrapezius or cremaster, hamster retractor, rat gracilis and rabbit and cat tenuissimus muscles)11,12 The microvasculature of thicker muscles like the ones of the murine hind limb could not be completely visualized with sufficient detail resolution In short, there is a considerable demand for high-resolution vascular 3D imaging techniques, which allow visualizing the microvasculature of thicker muscles

3D imaging techniques like micro-computed tomography (microCT) have been receiving increasing attention

in recent years Despite providing volumetric data at near-microscopic resolution, the vasculature cannot be dis-criminated from other soft tissues on the basis of its inherent x-ray attenuation Thus, the vascular perfusion with

a radiopaque contrast agent is indispensible for visualizing the vasculature by microCT13 In vascular research, microCT combined with various perfusion-based contrast agents has been used to visualize the vasculature of various organs, such as the heart, the liver, kidneys, lungs, the hind limb but also of tumors14–20 However, those studies were limited in terms of resolution, size of volume of interest, filling and perfusion of the vasculature (also due to the relatively high viscosity of the applied perfusion-based contrast agent)13,17 and, therefore, restricted to the detection of vessels sized 20–50 μ m in the diameter In a recent publication by Ehling J and coworkers20 The authors stated that “blood vessels as small as 3.4 μm …could be visualized…” Unfortunately, declared isotropic voxel sizes ranging from 3.4 to 5.3 µm do not allow the unambiguous identification of such small vessels - not even in theory

Vascular corrosion casting followed by tissue maceration provides better detectability and has been success-fully used for microCT-visualization of capillaries in a tiny subsample, e.g., an individual glomerulus (scanned at

an isotropic voxel side length of 1 µm)21 Although several groups have demonstrated the feasibility of visualizing capillaries in skeletal muscle when using vascular corrosion casts, to our knowledge, no one has yet succeeded in

visualizing capillaries in situ by microCT, without prior tissue maceration21–24

In this study, we introduce a correlative ex vivo imaging approach, which allows investigating the murine

hind limb vasculature and its surrounding tissue from the whole organ to the capillary level To visualize the vas-culature, we used the novel polymerizing vascular contrast agent μ Angiofil® , which provides a sufficient strong signal to depict the vasculature including capillaries In addition, to visualize the muscle fibers, the tissue was dehydrated and covered with a thin layer of paraffin Finally, the tissue was further processed for histology and/or immunohistochemistry, which enabled a detailed characterization of the microvasculature of any region of inter-est In order to exemplify the utility of this approach, we applied it to analyze the angiogenic effects induced by the local overexpression of a controlled dose of Vascular Endothelial Growth Factor-A (VEGF) in skeletal muscle, taking advantage of a well-characterized cell-based gene delivery platform25,26

Methods

For a detailed description of the experimental methods, please see the Materials and Methods section of the Online Data Supplement

Myoblast implantation To study the local effects of VEGF overexpression, a previously described mono-clonal population of primary murine myoblasts was implanted in three hind limb muscles of SCID CB.17 mice,

as previously described27,28 The animals were housed and sacrificed according to the Swiss Animal Welfare Act and Swiss Animal Welfare Ordinance All experimental protocols were approved either by the veterinary office of the canton of Bern with the license number BE27/12 or the veterinary office of the canton of Basel-Stadt with the license number 2071 In total 11 mice were used in the study (n = 6 for cell implantation and n = 5 for establishing the method/non-injected control)

Sample preparation For the visualization of the vasculature Deeply anaesthetized mice were injected

with μ Angiofil® (Fumedica AG, Muri, AG, Switzerland), a polymerizing vascular contrast agent, through the descending aorta After polymerization, hind limbs were collected, fixed in 2% paraformaldehyde and stored until scanning

For the visualization of the muscle fiber architecture Samples were prepared as described above Before

scan-ning, hind limbs were decalcified over 4 days in 10% EDTA (pH 7.5), dehydrated and covered with a thin layer

of paraffin

MicroCT imaging Samples were scanned using a desktop microCT (SkyScan 1172 or 1272, Bruker, MicroCT, Kontich, Belgium) MicroCT projections were back projection-reconstructed using the NRecon soft-ware (NReconServer64bit, Bruker, MicroCT, Kontich, Belgium) and volume-rendered and visualized in 3D with the CTVox software (Bruker, MicroCT, Kontich, Belgium) Muscle tissue and blood vessels were segmented and analyzed using the CTAn software (Bruker, MicroCT, Kontich, Belgium) Blood vessel sizes were assessed using Matlab (The MathWorks, Inc., Natick, MA, USA) and plotted using Excel (Microsoft Corporation, Redmond,

WA, USA)

Histology, immunohistochemistry and immunofluorescence After microCT scanning, decalcified hind limbs or single muscles were further processed for histology 5 μ m-thick paraffin-sections were prepared and stained either with Azan trichrome, Masson trichrome, BS-1 Lectin (L3759, Sigma Aldrich Co., St Louis, MO, USA) or with a monoclonal anti-slow skeletal myosin heavy chain antibody [NOQ7.5.4D] (Abcam, Cambridge, UK) Azan and Masson trichrome were chosen to facilitate the comparability between histology and microCT: both stainings highlight the connective tissue (appearing in blue), which also appears lighter colored in x-ray projections (higher x-ray attenuation than muscle tissue)

Perfusion efficiency Perfusion efficiency was assessed based on lectin-stained histological sections, where

μ Angiofil® -perfused and non-perfused vessels were determined Using a systematic random sampling proce-dure 4 fields of view were selected per section and 3 sections per muscle Therein, a total of 1265 ± 88 capillaries per animal were evaluated by classifying each capillary either as μ Angiofil-positive/Lectin-positive (μ AF+ /L+ ),

μ Angiofil-positive/Lectin-negative (μ AF+ /L− ), or μ Angiofil-negative/Lectin-positive (μ AF− /L+ )

Density maps Density maps representing the number of capillaries per area (capillary density) and the capillary-to-fiber ratio were generated For the former one, blood vessels were manually identified in the digital image and analyzed using an in-house written Matlab-script For each kernel (squared area) the blood vessels were summed up and the sum (number of blood vessels contained in a kernel) was eventually divided by the area

of the kernel (in μ m2) For the latter one, the capillary-to-fiber ratio within a pre-defined area (kernel) was deter-mined and the density map was generated

Results Visualization of blood vessels and muscle fibers/muscle fiber bundles For the consecutive imag-ing of blood vessels and their surroundimag-ing muscle tissue we established a multi-stage procedure (Fig. 1A) First, the blood was removed from deeply anaesthetized and heparinized mice Thereafter, approximately 3 ml of the dark blue μ Angiofil® were injected into the circulation via the descending aorta As soon as μ Angiofil® polymer-ized, hind limbs were collected, muscles were dissected, fixed and stored (for days or weeks) until being scanned The first microCT-scan was performed in order to visualize the contrast-enhanced vasculature (Fig. 1B,C)

In a next step, the muscle was prepared similar to the standard procedure for histology: it was dehydrated in an ascending alcohol series, and then transferred to xylene substitute before being infiltrated by paraffin However, instead of embedding the muscle into a paraffin-block, only a thin layer of paraffin was left around the muscle

At this point, a second microCT-scan was performed to delineate muscle fibers (Fig. 1D,E) The most compelling scanning parameters that were used for the different applications were summarized in Table 1 Lastly, the muscle was embedded for histology, sectioned and stained (Fig. 1F) This three-step protocol used microCT to provide 3D morphological information of the vasculature and the musculoskeletal system and standard immunohisto-chemistry to generate complementary 2D information at the microscopic level

At each of these steps, tissue shrinkage occurred to a different extent (Fig. 1G) The relative shrinkage at each

of the aforementioned steps was assessed by comparing microCT-derived volume measurements (relative volume shrinkage) or muscle area measurements (relative area shrinkage) From the unfixed muscle to its fixed state (= microCT-scan of the vasculature) no volume shrinkage was observed (109%), whereas from the fixed to the dehydrated/paraffinized state (= microCT-scan of the muscle fibers) considerable shrinkage of 63% was measured The area shrinkage between the dehydrated and the sectioned state (= histology) was 1% Consequently, it was not possible to directly co-register blood vessels and muscle fibres; still, they can be co-registered regarding their level and obliquity Although isotropic shrinkage occurred, no significant tissue distortion was noticed, suggesting that the morphological structures kept their relationship to each other

Musculoskeletal system of the murine hind limb For a better orientation within the murine hind limb, we performed an overview scan of the entire musculoskeletal system After 3D reconstruction, virtual sec-tions through the entire hind limb could be obtained at any given height and in any given plane (sagittal,

longi-tudinal or transversal), without destroying the sample (Fig. 2A–C and online video S1) Because connective tissue

components showed slightly higher x-ray attenuation than the muscle fibers themselves, individual muscles were readily identifiable Even at a relatively low resolution (isometric voxel side length: 2.99 μ m), muscle fibers bun-dles were distinguishable and their orientation and fiber architecture including pennation angles could be clearly delineated Moreover, since the sample remained intact, it was further processed for histology (Fig. 2C’)

Contrast-enhanced microvasculature of the murine hind limb visualized by microCT In order

to study the vascular network of the murine hind limb in 3D, a multi-scale imaging approach was followed First,

an overview scan of the contrast-enhanced vasculature was acquired (Fig. 3A,B) This enabled the visualization

of all blood vessels 10 μ m in diameter or larger The intravital injection of the solidifying contrast agent enabled

an accurate visualization of the vasculature in situ and preserves the spatial relationship to other

morpholog-ical structures, such as bones and nerves Two muscles, soleus and plantaris, known to exhibit different fiber type characteristics, were chosen to further characterize their microvascular architecture Both muscles were scanned at higher resolution (voxel side length: 0.8 resp 0.66 μ m) to detect all blood vessels including capil-laries (Fig. 3C–F) Based on virtual transverse sections of the two muscles, equally sized volumes of interest were selected in order to study the vascular pattern in more detail (Fig. 3C’–F’) At higher magnification, the differences in vascular pattern between different muscles became more evident In soleus muscles, capillaries

appeared highly tortuous and built a dense vascular network surrounding the muscle fibers (Fig. 3C’,D’ and online video S2), whereas in plantaris muscles, capillaries appeared straighter, more sparsely arranged and less organized

(Fig. 3E’,F’)

This multi-scale top-to-bottom imaging approach provides 3D information on the microvasculature and, at the same time, enables the maintenance of an overall view of the entire hind limb

Perfusion efficiency of μAngiofil ® To assess the perfusion efficiency of μ Angiofil® , scanned muscles were further processed for histology Serial sections were prepared and imaged at high magnification Because

of the μ Angiofil® ’s strong inherent fluorescent signal, perfused blood vessels were detected without prior vessel staining Subsequently, lectin-stained histological sections were manually registered across the entire microCT dataset to find their corresponding virtual sections The comparison of these section pairs, showed an excellent

agreement between the two imaging modalities (Fig. 4A,B) In a next step, the perfusion efficiency of μ Angiofil® was quantitatively assessed comparing μ Angiofil® -induced auto-fluorescence with lectin staining (Fig. 4B,C) A total of 3800 capillaries were evaluated (Fig. 4D) 91% (± 3%) of all capillaries were μ Angiofil® - & lectin-positive,

Figure 1 Correlative imaging approach to visualize the vasculature and fiber arrangement by microCT and histology (A) Workflow (B,C) Contrast-enhanced vasculature of plantaris muscle in 2D (B) and 3D (C) Voxel side length: 0.99 μ m (C,D) Muscle fibers of the same plantaris muscle in 2D (C) and 3D (D) Voxel side length: 1.39 μ m (F) Corresponding histological cross-section stained with Masson trichrome (G) Estimated

shrinkage between the different steps of the procedure Larger blood vessels were manually highlighted in white

2% (± 0.5%) were only lectin-positive, whereas 7% (± 3%) were only μ Angiofil® -positive μ Angiofil® allowed an adequate perfusion and visualization of the entire vasculature including the smallest capillaries

Heterogeneity of skeletal muscle capillarization Skeletal muscle is composed of different muscle fiber types which can be classified into slow and fast-contracting muscle fibers29 While some muscles display a rather homogeneous mosaic of fiber types, others display enormous regional heterogeneities Consequently, capillaries are not evenly distributed throughout such muscles Thus, two density maps based on histological images were generated to evaluate the spatial distribution of capillaries throughout gastrocnemius medialis muscle because

it is known to be composed of a mixture of slow- and fast-twitch fibers30,31 (Fig. 5) Figure 5A shows a “con-ventional” density map representing the number of capillaries per area, whereas Fig. 5B displays the relative distribution of capillaries and fibers (capillary-to-fiber ratio) Both, capillary density and capillary-to-fiber ratio distribution indicated a highly vascularized deep muscle portion and a sparsely vascularized superficial portion

As expected, this correlated well with the distribution of slow muscle fibers, which were stained using an anti-slow skeletal myosin heavy chain antibody (Fig. 5C)

Effects of site-specific delivery of VEGF-transfected myoblasts We sought to further validate this novel vascular imaging approach by performing a 3D analysis of VEGF–induced vascular growth For this pur-pose, we took advantage of a previously well-characterized model of controlled VEGF gene delivery for ther-apeutic angiogenesis in skeletal muscle This platform relies on monoclonal myoblast populations retrovirally transduced to over-express specific and homogeneous levels of VEGF, allowing a localized, sustained and con-trolled VEGF-overexpression with no systemic effects26–28 A myoblast clone homogeneously secreting a moder-ate amount of VEGF, which was previously shown to robustly induce only normal and therapeutically effective angiogenesis25,32, was chosen and implanted into 3 hind limb muscles of immunodeficient SCID mice (n = 6) CD8+-control-myoblasts (CD8-Ctrl) were injected into the contralateral hind limb instead Since it was pre-viously shown that networks of normal and mature capillaries are fully formed 1 week post implantation of the VEGF-expressing myoblasts25, hind limbs were collected and scanned using microCT 10 days post implantation Based on the overview scans (voxel side length: 2.58 μ m), no significant vascular effects could be detected in any

of the control hind limbs (Fig. 6A,D), whereas the three injection sites of VEGF-transduced myoblasts could be clearly identified and localized, and envelopes of densely arranged vessels could be seen surrounding each

injec-tion site (Fig. 6D and online video S3).

As expected, vascular volume measurements of the injection sites indicated robust vascular growth in the VEGF-treated muscles compared to CD8-Ctrl and uninjected muscles (0.32 ± 0.13 vs 0.08 ± 0.03 and 0.12 ± 0.05,

p < 0.0001) (Fig. 6G) Muscles treated with CD8-control cells did not show any vascular changes compared to the non-treated control muscles

In order to obtain a more detailed view of the injection sites and their surrounding tissue, the soleii muscles were carefully isolated The high-resolution scan (voxel side length: 0.92 μ m) revealed a dense vascular network comprising tortuous and enlarged blood vessels, but with a regular morphology, in close vicinity to the injection site (Fig. 6E,F) Quantification of vessel size distribution (Fig. 6H) confirmed an increase in diameter in the VEGF-treated muscles compared to the control muscles (15.7 ± 1.7 (MedianVEGF) vs 13.4 ± 0.5 (MedianCD8) and 12.9 ± 0.6 (Medianuninjected)) As anticipated27,33, in this area immediately adjacent to the injection site no increase

in capillary-size structures could be measured, which are known to be induced exclusively in the microenvi-ronment around VEGF-expressing cells26 However, specifically medium-sized vessels larger than 20 μ m were increased, which was compatible with the previously described arteriologenic effect of VEGF27,33

Discussion MicroCT-based visualization of the skeletal muscle vasculature In this study, we present an ex vivo

microCT imaging approach, in combination with the novel polymerizing contrast agent μ Angiofil® , as a versatile

Tissue Fixation Pretreatment

MicroCT parameters

Scan duration MicroCT Visualization of

kV uA rotation step [°] filter

voxel side length [μm]

Vasculature

single muscle 2% PFA none 49 200 0.15-0.23 no 0.66-0.8 4h40 (1) 1172 microvasculature incl capillaries

Musculoskeletal system

connective tissue

Application

control myoblasts

Table 1 Sample preparation and scanning parameters *Enclosed in brackets you find the number of

segments that were scanned

and widely applicable tool for vascular-related research Although microCT has been frequently used to evaluate arteriogenesis/collateral vessel formation, no one has yet succeeded in visualizing the vasculature in its entirety, down to the capillary level34–37

To our knowledge, we are the first to present microCT-based visualization of skeletal muscle’s vasculature down to capillary level without prior tissue maceration This microCT approach provides detailed tomographic 3D data at near-microscopic resolution, thereby providing new insights into the microvascular architecture, its hierarchical structure and geometrical complexity, such as branching pattern, anastomoses and tortuosity As demonstrated, the capillaries within soleus muscle are much more tortuous and build more anastomoses than the ones in plantaris muscle Few researchers considered the heterogeneous distribution of blood vessels within muscles when evaluating the vasculature by classical histology To overcome potential sampling bias, which could lead to erroneous results, we suggest quantifying vascular volumes instead

Figure 2 Musculoskeletal system of the left murine lower hind limb (A) Lateral, (B) Sagittal and (C) Transversal view through the hind limb imaged by microCT Voxel side length: 2.99 μ m (C’) Corresponding

histological cross-section stained for Azan Trichrom (blue: bones and connective tissue, red-yellow: muscle

tissue (C”) Scheme with labelled muscles T = tibia, F = fibula, TA = tibialis anterior, EDL = extensor

digitorum longus, EHL = extensor hallucis longus, PB = peroneus brevis, PL = peroneus longus, FDL = flexor digitorum longus, TP = tibialis posterior, FHL = flexor hallucis longus, SOL = soleus, Plant = plantaris, Gc med = gastrocnemius medialis, Gc lat = gastrocnemius lateralis

Furthermore, this data could be particularly useful for the creation of computational models to improve simulations on blood flow and shear stress as well as facilitate progress in the field of microvascular network analysis While earlier studies dealing with microvascular network analysis were mainly restricted to the analysis of flat and easily accessible tissue samples and/or thin muscles of the animals that could be transilluminated11 The here presented approach provides 3D-datasets of significantly thicker tissue samples enabling a qualitatively new level

of microvascular network analysis

Figure 3 Vasculature of the murine lower hind limb visualized by microCT (A) Lateral 3D view of the hind limb vasculature Voxel side length: 2.7 μ m (B) Virtual transverse section of the vasculature (at the level indicated in A) Tibia (T) and fibula (F) appear lightly colored due to their high x-ray absorption (C–F) Virtual transverse sections of isolated soleus (C,D) and plantaris (E,F) muscle, respectively Voxel side length: 0.8 and 0.66 μ m respectively (C’–F’) Volumes of interest indicated by rectangles in (C–F), showing volume-rendered

microvasculature at higher magnification with differing vessel densities, tortuosity and 3D arrangement

Benefits Manpower and time efficiency Current approaches for 3D visualization of blood vessels include

serial sectioning or vascular corrosion casting, both of which are destructive, time-consuming and entail various difficulties Unlike these methods, microCT allows time-efficient 3D imaging without loss of any sections Taken together, sample preparation (30 min), fixation (overnight), image acquisition (4 h), reconstruction (approx 4 h), post-processing (2 h) and generation of the final 3D image, all can be performed within 2 days

Correlative morphology MicroCT is a non-destructive imaging technique, so upon scanning, the chemically

fixed tissue is still amenable for further analyses by light (or electron) microscopy Therefore, we developed a correlative imaging approach, combining microCT with subsequent histology This approach provides consider-ably more information than “blind” serial sectioning and enables us to visualize structures from different levels

of organization, from whole organ down to cellular level Furthermore, since the used immunohistochemical protocol yielded good results, we do not see any apparent obstacles for the whole-mount staining It makes our suggested approach even more promising by enabling comparison of multiple 3D imaging modalities Altogether, complementary information of any region of interest can be extracted and gathered in a very effective and reliable way

μAngiofil® - a new generation contrast agent The microCT-based visualization of blood vessels relies heavily

on the contrast agent used to enhance the otherwise low inherent contrast of blood vessels Most of the available perfusion-based contrast agents are based on either iodine or heavy metals put in suspension High toxicity, clumping and vessel obstructions, inhomogeneous signal and disconnected vessels are common issues that have

Figure 4 Validation of microCT data by standard histology (A) Virtual cross-section of the vasculature

of soleus muscle obtained by high-resolution microCT Voxel side length: 0.8 μ m (B,C) Corresponding

histological cross-sections Vessels filled with μ Angiofil® (green) (B) and subsequent section stained for lectin (brown) (C) (A’,B’,C’) Region of interest, indicated with squares, at higher magnification (D) Perfusion

efficiency (n = 3)

been described24 In this study, we used the recently developed perfusion-based contrast agent μ Angiofil® , which has proven to be an excellent contrast enhancer with extraordinary perfusion properties, yielding a homogeneous signal throughout the tissue specimen It enters and fills even smallest blood vessels, thus ensuring adequate per-fusion of the entire microvascular network Moreover, it solidifies and retains the vessel morphology but remains elastic enough to allow for easy post-dissection of the tissue of interest Furthermore, samples were stored in 2% PFA over several months before being scanned and the contrast remained stable Hence, as long as the samples are not physically destroyed through sectioning, they can be rescanned almost any time Another convenient feature

of μ Angiofil® is its inherent fluorescence, which renders tedious capillary staining for fluorescence microscopy unnecessary

Limitations Perfusion pressure Similar to other perfusion techniques, special care should be taken to keep

the perfusion pressure as uniform as possible across the different animals Still, the pressure probably exceeded the physiological one, which could lead to unwanted vessel dilation or leakage By gross examination we did not find any vessel leakage in any of the non-treated mice and blood vessel sizes were in the normal range of what had been described in the literature10 Nevertheless, absolute values for vessel diameters must be interpreted with caution

Tissue shrinkage Another critical issue, usually underestimated or neglected, is tissue shrinkage occurring

during sample preparation for microCT imaging Only few studies have addressed this important issue9,38,39 Consistent with what had been described previously, we observed an initial swelling of the muscle which was then followed by substantial shrinkage mainly during the dehydration with the increasing alcohol series38 Similarly,

Buytaert et al found volume shrinkage of nearly 60% after having applied an ethanol-based staining procedure to

enhance skeletal muscle’s contrast40 Despite this, corresponding sections could be easily identified suggesting that shrinkage occurred homogeneously throughout the sample without any significant deformation

Post-mortem microCT application The reported imaging modality is designed exclusively for post-mortem analyses and does not allow dynamic 3D monitoring of vascular alterations in vivo (longitudinal studies) Although in vivo microCT-scanners are constantly improving, the repeated x-ray irradiation exposure and the

low spatial resolution remain yet unsolved problems MicroCT provides purely structural information, and does not provide any functional data There are other imaging modalities that could provide some functional data, such as Laser Doppler imaging, ultrasound imaging (microbubbles) or contrast echocardiography, positron emis-sion tomography (PET) or single-photon emisemis-sion computed tomography (SPECT)41–43 However, the spatial resolution of these imaging technologies is still poor, sensitivity and penetration depth limited

Data storage Further limitations of high-resolution 3D imaging include the tremendous amount of data that is

generated, especially when high-resolution scans of multiple segments are needed Hence, image storage, transfer, display and analysis are major challenges and require very powerful computers

Figure 5 Capillary density correlates with slow muscle fiber distribution in the medial gastrocnemius muscle (A) Density maps representing number of capillaries per area (capillary density) (A) and capillary-to-fiber ratio (B) (C) Slow muscle capillary-to-fibers distribution revealed by anti-slow skeletal myosin heavy chain antibody.

Figure 6 Therapeutic application of VEGF-transduced myoblasts (A,D) Vascular microCT-scans of CD8-control (A) and VEGF-treated (D) contralateral hind limbs respectively (voxel side length: 2.58 μ m) VEGF-injection sites are indicated by arrows (B–F) High-resolution microCT-scans of the soleii muscles (B,E) and regions of interest at higher magnification (C,F) (voxel side length: 0.92 μ m) (G) Vascular

volume measurements Vascular volume (vv) per tissue volume (tv) within a given injection or control site

****p < 0.0001 (H) Vessel size distribution Histogram presenting the relative proportion of a given vessel size

Vessel diameters are given in μ m Measurements were conducted on 20 equidistant virtual sections per sample